The objective of this study is to elucidate the intracellular interactions of the HIV regulatory protein Nef with host cell activities. Early in the HIV-1 infection of CD4 T cells this retrovirus expresses regulatory proteins, with the Nef transcript representing nearly 80% of total viral mRNA. In vivo infectivity by SIV and HIV is achieved in the absence of Nef expression, but there is an absolute requirement for Nef in the production of high viral titers. In the absence of Nef, there is loss of development of the immunological and neurological dysfunctions characteristic of AIDS. The essential activity of the Nef protein in the development of AIDS has not been defined. We have discovered that expression of Nef in human CD4 T cells results in a lowering of the activation threshold. Since activation is essential for viral synthesis, Nef thus has a direct role in increasing viral production. Evidence suggests that the biochemical site affected by Nef is early in the T cell receptor pathway with down-stream enhancement of the Erk cascade. We had previously established that the Nef protein associates with a member of the p21-activated kinase (PAK) family, which is known to initiate activation pathways as well as alter cytoskeletal structures. Our exploration of early infection of HIV in quiescent CD4 T cells has lead to the discovery that prior to integration, reverse-transcribed HIV DNA generates multiply spliced transcripts that encode the HIV regulatory proteins. These expressed proteins prime quiescent cells for activation and enhanced viral production. NeuroAIDS is characterized by neuronal death, although HIV does not infect neurons. Infection is largely restricted to the peripheral monocyte-derived brain macrophages and microglia. These cells are responsible for antigen presentation and immunological functions in the brain. There is limited knowledge of the role of Nef in macrophage and microglia, although there is evidence that Nef contributes to proinflammatory processes. It is presently believed that this inflammation, along with other secreted HIV gene products, contributes to neuron death. Infection of macaques by Nef-negative SIV results in no pathology--including no brain disease. We are presently examining biochemical pathways in macrophage and microglial cells that are parallel to those affected by Nef expression in T cells. These pathways in macrophages and microglia are responsible for secretion of inflammatory cytokines and chemokines. An understanding of the biochemical activity of this virulence factor could lead to new options for treating HIV-infected individuals.